1. Cell Division in Development, Adult Tissue Homeostasis, and Disease
Metazoan cell division involves dramatic changes of cellular structures. Both secretion and endocytosis are either greatly slowed down or stopped as the cell prepares for mitosis [1-3]. As mitotic spindle assembly progresses and chromosomes further condense [4], the dissolution of nuclear envelope and nuclear lamina [5] is accompanied by the fragmentation of Golgi ribbons [6] and changes in other membrane compartments including the endoplasmic reticulum [7], the endosomes [3], and the mitochondria [8]. Therefore, successful mitosis requires not only an equal segregation of chromosomes into daughter cells but also proper partitioning of other cellular components such as the membrane systems.
Proper cell division is fundamentally important in normal development and tissue homeostasis. For example, equal chromosome segregation during cell division is required for maintaining genome stability [4]. Correct segregation of nuclear materials, including transcription factors, is essential for the daughter cells to reestablish the interphase nuclei with appropriate transcription programs that allow them to either progress along an appropriate differentiation pathway or to remain undifferentiated (as in the case of division of progenitor and stem cells) [9]. Moreover, proper partitioning of the membrane system is necessary to ensure that each of the daughter cells can reestablish both endocytic and exocytic pathways that allow them to send and receive signals and to coordinate organogenesis and tissue homeostasis [10].
Inappropriate cell division could lead to defects in development and adult tissue homeostasis, which results in disease. Indeed, many human diseases, including cancer, premature aging, disease of the vasculature and the airway, and diabetes, are directly related to problems in cell division, differentiation, and cell death. For example, cancer is a result of uncontrolled cell division. Premature aging such as certain types of laminopathy is largely caused by lack of cell proliferation. Increased proliferation and decreased apoptosis of smooth muscle cells contribute toward airway obstruction in asthma, atheroma and restenosis after vascular injury.
2. The Role of Mitotic Spindle Morphogenesis in Cell Division and Differentiation
The assembly of the microtubule (MT)-based bipolar spindle apparatus is essential for cell division. One of the important functions of the spindle apparatus is to capture the condensed chromosomes on the MT-based spindle fibers. Equal chromosome segregation requires that each sister chromatids to be captured by spindle fibers originated from opposite spindle poles. An elaborate surveillance system called spindle checkpoint has evolved to monitor the capture of chromosomes by the spindle fibers. The spindle checkpoint senses inappropriate chromosome capture and is able to arrest mitosis until all chromosomes have achieved correct attachment to the spindle.
Another important function of the spindle apparatus is to regulate cell differentiation during development. The position and geometry of mitotic spindle can determine the plane of cell division. Studies in multiple systems have shown that cell-cell signaling regulates spindle positioning and spindle shape. The mitotic spindle in turn functions to differentially partition cell fate determinants into daughter cells. Through differential interaction with the membrane systems, the mitotic spindle also actively segregate signaling molecules into one but not the other daughter cells [11].
3. The Mitotic Spindle as a Target of Therapeutic Interventions and its Current Limitations
The importance of spindle morphogenesis in maintaining genome stability and in ensuring proper cell differentiation makes it an ideal target for therapeutic interventions of many diseases. For example, chemicals such as taxol that disrupts proper MT polymerization have been developed to treat cancer [12]. Additional chemicals that disrupt either MT-based motor proteins [12] or kinases that regulate cell division, such as CDK kinases [13], Polo-like kinase 1 [14], and Aurora A and B kinases [15] are at different stages of development or clinical trials. Extensive studies have shown that many of these chemicals can cause prolonged cell division block and initial tumors regression. However, a number of outcomes of such treatments eventually render additional genomic instability and tumor re-growth as some tumor cells escape the prolonged mitotic arrests, survive and continue to undergo cell cycle [16]. The limited success of anti-cancer drugs calls for the development of additional chemicals that can both arrest tumor cell division and cause cell death. However, the current understanding of spindle morphogenesis has put a significant constraint on the development of assay systems to identify such kind chemicals.
Differentiation of stem cells into different tissues holds great promise in the treatment of various human diseases. The ability of mitotic spindle to orchestrate differential partitioning of cell fate determinants offers a great potential to identify compounds that could induce lineage specific tissue differentiation from stem cells cultured in vitro. However, the lack of understanding of how the mitotic spindle interacts with cell fate determinants has made it difficult to design strategies to identify such compounds.
4. The Identification of Mitotic Spindle Matrix Presents a Conceptual Advancement of Understanding the Role of Spindle Morphogenesis in Cell Division and Cell Differentiation
Mitotic spindle assembly and chromosome segregation is a dynamic and force production process, which requires coordinated actions of MTs, MT-based motors, MT-binding proteins, and chromosomes [17, 18]. Although much progress has been made in understanding how the MT-based spindle regulates chromosomes segregation, it has become apparent that proper spindle morphogenesis and cell division involves additional intracellular structures besides the MT cytoskeleton. Indeed, it was hypothesized decades ago that a static scaffold, called the spindle matrix, exists during mitosis and is required for cell division. Such a matrix could tether spindle assembly factors (SAF) to support the assembly and force production of spindle microtubules [19-21]. However, the existence and molecular nature of the spindle matrix had remained elusive until our recent discoveries, which are disclosed in this invention.
Previous studies have shown that the guanosine triphosphatase (GTPase) Ran, a protein with well-established function in interphase nuclear trafficking, plays an important role in regulating spindle morphogenesis in mitosis [22-29]. Moreover, it was determined that RanGTP functions in a signaling pathway that leads to the activation of the mitotic kinase Aurora A [30, 31]. Based on these findings, a number of assays have been devised that allowed identification, biochemical isolation and characterization of the mitotic spindle matrix [32, 33].
As disclosed in this invention, it was determined that RanGTP induces the assembly of the mitotic spindle matrix, which is essential for spindle morphogenesis [33]. It was demonstrated demonstrated that the mitotic spindle matrix associates with spindle microtubules and consists of a membrane system and the polymerized nuclear lamin B. Moreover, this matrix tethers a number of spindle assembly factors that are known to promote both microtubule nucleation and organization. Finally, the mitotic spindle matrix contains transcription factors and signaling molecules known to regulate cell proliferation, stem cell pluripotency, and cell differentiation. These discoveries provide an important conceptual advancement that explains how mitotic spindle morphogenesis is required for not only chromosome segregation but also for the partitioning of cell fate determines and membrane systems into daughter cells. Therefore, the mitotic spindle matrix identified in this invention differs significantly both in its functions and contents from the originally hypothesized spindle matrix. The mitotic spindle matrix is also distinct from the interphase nuclear lamina in its structure, composition, function, and the requirement of the mitotic state for its assembly.
5. The Ability to Prepare and Isolate the Mitotic Spindle Matrix Offers Distinct Approaches to Assay for Cell Division and Differentiation
The characteristics of the mitotic spindle matrix disclosed in this invention allow us to devise methods (also disclosed in this invention) to prepare and isolate the mitotic spindle matrix. These methods are conceptually and practically distinct from the existing methods of isolating mitotic spindles. The existing spindle-isolation methods involve the removal of DNA and RNA and the utilization of detergents [34], which prevent the isolation of a fully functional mitotic spindle matrix. By modulating the mitotic spindle matrix, our invention offers distinct strategies to manipulate cell division and differentiation potentials that are not currently available.